Objective: To investigate the protective effect of Danshenxinkun B against oxidized low-density lipoprotein (ox-LDL)- induced human umbilical vein endothelial cell (HUVEC) injury and explore the underlying mechanism.
Methods: HUVECs cultured in the presence of 10% fetal bovine serum were treated with ox-LDL (100 μg/mL), ox-LDL+0.1% dimethyl sulfoxide (DMSO), or ox-LDL+Danshenxinkun B (100 ng/mL, dissolved in DMSO) for 24 h. The changes in lactate dehydrogenase (LDH) release was detected, and qRT-PCR was used to detect the mRNA expressions of nuclear factor-κB1 (NF-κB1), nucleotide binding oligomerization domain-like receptor family pyrin domain protein 3 (NLRP3), gasdermin D (GSDMD) and interleukin- 1β (IL-1β). The protein expressions of NF-κB1, NLRP3, caspase-1, IL-1β and GSDMD-N were detected with Western blotting. Immunofluorescence assay was performed to examine the changes in GSDMD expression in the cells.
Results: Compared with the normal control cells, the cells treated with ox-LDL alone or in combination with DMSO exhibited significantly increased LDH release, mRNA expressions of NF-κB1, NLRP3, GSDMD, and IL- 1β and the protein levels of NF-κB1, NLRP3, IL- 1β, GSDMD-N and caspase-1 (P<0.01), which were all significantly lowered by treatment with Danshenxinkun B (P<0.05 or 0.01). Danshenxinkun B treatment significantly inhibited GSDMD expression on the cell membrane and restricted its entry into the cell nucleus.
Conclusion: Danshenxinkun B alleviates ox-LDL-induced HUVEC injury possibly by suppressing pyroptosis mediated by NLRP3 inflammatory bodies and inhibiting the NF-κB/NLRP3 signaling pathway.
目的: 探讨丹参新醌乙对氧化低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞损伤的影响及机制。
方法: 取对数生长期的人脐静脉内皮细胞，分为正常组(加入10%胎牛血清)、ox-LDL组(在正常组基础上加入100ug/mL的ox-LDL)、二甲基亚砜(DMSO)组(在ox-LDL组基础上加入0.1%浓度DMSO)、丹参新醌乙组(在ox-LDL组基础上加入由DMSO溶解的100ng/ml的丹参新醌乙)，培养24 h后进行实验。微板法检测乳酸脱氢酶(LDH)表达；qRT-PCR法检测NF-κB1、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、消皮素D(GSDMD)、白介素(IL)-1β的mRNA表达；Western Blot法检测NF-κB1、NLRP3、caspase-1、GSDMD-N的蛋白表达。免疫荧光法检测GSDMD表达情况。
结果: 与正常组比较，ox-LDL组、DMSO组LDH表达明显升高(P<0.01)，NF-κB1、NLRP3、GSDMD、IL-1β的mRNA水平均显著上调(P<0.01)，NF-κB1、NLRP3、caspase-1、GSDMD-N、IL-1β的蛋白表达水平明显升高(P<0.01)；与ox-LDL组、DMSO组比较，丹参新醌乙组LDH表达有所降低(P<0.05)，NF-κB1、NLRP3、GSDMD、IL-1β的mRNA水平显著下调(P<0.01)，NF-κB1、NLRP3、caspase-1、GSDMD-N、IL-1β的蛋白水平显著降低(P<0.01)，GSDMD在质膜表达抑制，入核受限。
结论: 丹参新醌乙可能通过NF-κB/NLRP3信号通路，抑制NLRP3炎症小体介导的细胞焦亡，改善ox-LDL诱导的内皮细胞损伤。
Keywords: Danshenxinkun B; NF-κB/NLRP3 signaling pathway; endothelial cell injury; inflammation; oxidized low-density lipoprotein; pyroptosis; therosclerosis.